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Author Notes:

Corresponding author: ddunlap@emory.edu, Tel: 404-727-3951.

We are grateful to Dr. Sunita Subramanian and Professor Graeme Conn in the Biochemistry Dept. at Emory University for help with the column chromatography.

Subjects:

Research Funding:

This work was supported by the Human Frontier Science Program (RGP0051/2009) and the National Institutes of Health (RGM084070A).

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemical Research Methods
  • Biochemistry & Molecular Biology
  • Biotechnology & Applied Microbiology
  • Lambda repressor
  • Bacteriophage lambda
  • Heparin affinity
  • Tethered particle motion
  • Ammonium sulfate
  • Anion exchange
  • Single particle tracking
  • His-tag
  • C-TERMINAL DOMAIN
  • COOPERATIVE OPERATOR BINDING
  • TETHERED PARTICLE MOTION
  • SINGLE-SITE MUTATIONS
  • ESCHERICHIA-COLI
  • DNA LOOPS
  • GENE-REGULATION
  • EXPRESSION
  • PROTEIN
  • AUTOREGULATION

Purification of bacteriophage lambda repressor

Tools:

Journal Title:

Protein Expression and Purification

Volume:

Volume 91, Number 1

Publisher:

, Pages 30-36

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy "footprinting" assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.

Copyright information:

© 2013 Elsevier Inc. All rights reserved.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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